The protein a-synuclein (aS) plays an important but poorly understood role in the pathogenesis of Parkinson's disease (PD). Mutations found in hereditary early-onset PD have been traced to the aS gene, ordered aggregates of aS are the primary component of the intracellular Lewy body deposits characteristic of PD, and mice and flies expressing human aS have provided the first transgenic animal models for PD. The normal function of aS remains unknown, but it is believed to be a synaptic vesicle-associated protein. aS undergoes a conformational change to a highly helical state upon interacting with lipid vesicles or SDS micelles, and this state is thought to represent the protein in one of possibly several normally functioning conformations. As is intrinsically unstructured when free in solution but slowly forms typical amyloid fibrils, similar to those that are found in Lewy bodies and are conjectured to play a causal role in PD. Residual structure in free aS may play an important role in mediating the intermolecular interactions that precede amyloid fibril formation, and the early-onset aS mutations may exert their pathogenic effects by modulating such residual structure. We propose to characterize, at high resolution, the structural and dynamic properties of aS in its free state using NMIR spectroscopy. We also propose to elucidate in detail the structure of SDS - and lipid vesicle-associated aS in order to gain insights into the normal structure and function of this protein. We will probe the effects of early-onset mutations (A3OP and A53T) and of newly designed aS mutations on structure and dynamics in its free and lipid-associated states. We will attempt to delineate and characterize specific sites involved in the initial oligomerization interactions that lead to aS fibril formation, and we will use mutants to test our conclusions, collaboratively, in a transgenic fly model of PD. The proposed studies are focused on improving our understanding of the molecular mechanisms underlying PD and may suggest strategies for developing new PD therapeutics. The results may have broader implications for understanding and treating other amyloid diseases, including Alzheimer's disease and the prion diseases.